西尼罗病毒RT-PCR检测方法的建立及其初步应用

目的　建立西尼罗病毒 (WestNilevirus ,WNV)RT PCR检测方法 ,为WNV感染的诊断和流行病学调查奠定基础。方法　选择Vero E6细胞进行WNV培养 ,通过乳鼠脑内接种病毒培养液获得WNV感染脑组织。在病毒基因组E区和C区设计 3对引物 ,以WNV培养液 ,建立并优化病毒核酸RT PCR分析方法。然后 ,以此对感染蚊虫模拟标本和乳鼠脑组织进行检测。PCR产物测序后 ,用Blast进行同源性分析。结果　用RT PCR在WNV培养液中扩增出与预期大小一致的核苷酸片段 ,并通过改变循环数 ,模板稀释倍数等参数对该方法进行了优化 ;将建立的方法用于检测感染蚊虫模拟标本和感染乳鼠脑组织 ,均检测出WNV目的基因片段 ,且扩增效果与病毒培养液无明显差异 ;套式PCR能显著提高检测的敏感性 ( 10 8-10 9倍 )。结论　本研究建立的WNVRT PCR检测方法具有较高的敏感性 ,可用于WNV感染的流行病学调查研究

Establishment and preliminary use of the RT-PCR technique for the detection of West Nile virus

To establish a RT PCR assay for the detection of West Nile virus (WNV) used as the laboratory diagnosis and epidemiological survey of the WNV infection, the Vero E6 cell was selected for the cultivation of WNV, and the WNV infected brain tissue was obtained by the intracerebral inoculation of viruses into suckling mice. Three sets of specific primers were designed in the E and C pre M regions of the viral genome, and the parameters of RT PCR amplification were optimized by using the template from the viral cultures. This method was used to detect WNV in infected brain tissues or the mimic samples of the infected mosquitoes Nucleotides of the PCR products were sequenced and analyzed by means of Blast program. It was found that the target DNA fragments could be amplified from the cell cultures infected with WNV by this RT PCR technique, and some PCR parameters, such as the cycles and dilution of template, were optimized. By using this established method of assay, the viral nucleotides could be identified in both the infected brain tissues and the mimic samples of infected mosquitoes. The nested PCR could remarkably increase the sensitivity up to 10 8 to 10 9 folds. It is concluded that the RT PCR established in the present study is a sensitive method for the detection of WNV, and can be used for the epidemiological survey on WNV infection.